The emergence of bacterial resistance to antibiotics continues to be a significant public health challenge. Factors contributing to the rise in antibiotic resistance include widespread and inappropriate prescription of broad spectrum antibiotics and patient non-compliance to antibiotic regiments. The healthcare community is responding by limiting antibiotics and prescribing targeted narrow-spectrum antimicrobial therapies when possible. Meanwhile, current antimicrobial development is focused more and more on narrow-spectrum drugs. To enable the effective use of antimicrobial therapy, new methods for detection of antibiotic susceptibility must be developed that enable physicians to prescribe these narrow spectrum antibiotics as close to initial examination as possible. Here, we propose to investigate a novel mechanism that would be the basis for a diagnostic to meet this important public health need. In this R21 application, Dr. Alexis Sauer-Budge at the Fraunhofer Center for Manufacturing Innovation at Boston University and Dr. Jean Lee of Brigham and Women's Hospital and Harvard Medical School have teamed up to explore the hypothesis that mechanical stress on bacteria will damage the cell wall and rapidly initiate cell wall repair biosynthesis pathways. In the presence of antibiotics targeted at cell wall biosynthesis, susceptible strains will be unable to recover from the damage and will die, whereas resistant strains will recover. The susceptibility of the bacteria can therefore be determined rapidly via fluorescence microscopy when the mechanical stress is applied in the presence of fluorescent dyes which stain the damaged bacteria. We propose to investigate this hypothesis by building a flow cell and optical apparatus, varying experimental conditions to optimize the protocol for antibiotic susceptibility detection, and quantify the variability due to different strains and growth phases of the bacteria. We propose to use the model system of Staphylococcus aureus, both methicillin-susceptible and methicillin- resistant strains. If successful, we anticipate this methodology will be applicable to a wide range of bacteria and antibiotics of various mechanisms of action. PUBLIC HEALTH RELEVANCE: The increasing prevalence of multi-drug resistant bacterial infections is a growing public health problem. To combat the trend and to fully enable physicians to prescribe appropriate antimicrobial therapy, new rapid diagnostic methods for antibiotic susceptibility must be developed. Here, we propose to address the gap in antibiotic susceptibility diagnostics by developing a novel technique for rapid detection of antibiotic susceptibility using Staphylococcus aureus as our model system.